This invention relates to increasing the uptake of gallium into cells for diagnostic and therapeutic purposes.
Gallium (Ga), a Group IIIa transition metal, has a number of isotopes with many medical uses. For decades, gallium-67, a gamma-emitter, has been used in nuclear medicine for tumor imaging by gamma emission scintigraphy (1). Currently, gallium-67 is most widely used in staging and assessing the therapeutic response of lymphomas (2, 3, 4, 5). Other isotopes of gallium have potential uses in oncology. Gallium-68, a positron emitter, can be used for tumor imaging by positron emission tomography (PET). Gallium-72, a beta-emitter, may destroy tissues that concentrate gallium by local radiation. This treatment has been proposed to palliate bone pain caused by skeletal metastases (6). Gallium-67 has also been used for local radiotherapy in the treatment of hematological malignancies (48, 49, 50, 51).
Stable (non-radioactive) gallium has been used to reduce the hypercalcemia of malignancy, and as a treatment for Paget""s disease of bone. It is also believed to have direct anti-neoplastic effects, and is currently under investigation as an adjunct to conventional chemotherapy (7, 8, 9).
The limitations of Ga-67 for oncologic imaging are well-recognized (10,11,12,13). Many tumors accumulate Ga poorly. Others, such as hepatomas and lymphomas, can be intensely Ga-avid but may vary in magnitude and consistency of uptake. Delineation of tumors from background tissues often requires extended intervals from the time of injection to imaging of 3-7 days or more because Ga-67 localizes slowly and initial images of the abdomen are frequently difficult to interpret because of bowel activity. Because of the extended intervals required for oncologic imaging, a relatively high dose of Ga-67 is required (typically 10 mCi for an adult). Despite its drawbacks, no other gamma-emitting radiopharmaceutical used for tumor imaging in nuclear medicine (including expensive monoclonal antibodies and receptor-avid peptides) has surpassed Ga-67 in cost-effectiveness, general availability, broad applicability and ease of imaging. Although efforts to improve the use of gallium are clearly justifiable, the techniques to accomplish this have thus far been elusive for impractical.
Despite years of imaging experience with the Ga-67 radiometal, the mechanism by which Ga-67 accumulates in normal tissues and tumors remains controversial. For years, it has been thought that gallium is taken up by cells as a gallium-transferrin (Ga-Tf) complex via the transferrin receptor (TfR) (14,15,16). However, there is also evidence that mechanisms other than the TfR may be responsible for the uptake of Ga-67 in tumors (17,18,19). For example, gallium may dissociate from Tf in the acidic extracellular environment of tumors, which would interfere with Tf mediated transport of cellular uptake (20, 21, 22). There is also a poor correlation between TfR density and the degree of tumor uptake of gallium. Moreover, gallium uptake continues to a significant degree even in the absence of Tf, or when TfR binding sites are blocked with an antibody or when iron overload down regulates TfR expression (23, 24, 25).
Tumor bearing rats that are rendered iron-deficient (which increases TfR""s in many tissues) exhibit an increased uptake of Ga-67 in tissue other than tumors (26). When Tf binding sites are saturated with iron or scandium after administration of Ga-67, uptake of gallium in tumors, relative to normal tissues, can actually increase (27, 28). Uptake of Ga-67 by nonosseous tissues and organs is markedly depressed in a hypotransferrinemic strain of mouse, suggesting that uptake of Ga-67 by most soft tissues and organs is a Tf-dependent process (29).
Nifedipine 1 (dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate) is a dihydropyridine calcium channel antagonist, which causes vasodilation and lowering of peripheral vascular resistance. These characteristics make nifedipine useful in the treatment of heart disease and hypertension. This compound, like most 1,4-dihydro-4-(2-nitrophenyl)pyridine derivatives, is very sensitive to light. Photo-degradation of nifedipine has been considered a drawback to its pharmaceutical use, because the photo-degradation products have been thought to lack pharmacological activity. Hence photo-degradation of nifedipine has diligently been avoided by shielding it from the light to prevent loss of its therapeutic properties. 
In the presence of light, nifedipine is converted to phenylpyridine derivative structures that include fully-aromatic compounds (FIG. 1). With exposure to visible/fluorescent light, nifedipine is converted predominantly to the 4-(2-nitrosophenyl)pyridine homologue 2 (the nitroso derivative, also known as 2,6-dimethyl-3,5-diacetyl-4-(2xe2x80x2-nitrosophenyl)-pyridine). When exposed to UV light, it is converted predominantly to the 4-(2-nitrophenyl)pyridine homologue 3 (the nitro nifedipine derivative). The nitro derivative is also the primary metabolic product of nifedipine in humans. In addition to these two main structures, photo-degraded nifedipine (PDN) also includes a broad variety of phenylpyridines such as the cis and trans-azoxy derivatives, the hydroxylamine derivative, the amine derivative, the lactam derivative, and the trans-N,Nxe2x80x2-dioxide derivative.
The present invention takes advantage of an unexpected property of nifedipine degradation products, such as photodegraded nifedipine products (PDN), or pharmaceutical analogs and their degradation products. This property can be used to improve the use of gallium for several purposes: 1) to improve tumor imaging; 2) to improve radiotherapy of tumors; and 3) to improve the use of gallium as an adjunct to chemotherapy. In particular example, the method can improve the uptake of gallium into tumor cells, to permit a total diagnostic or therapeutic dose of the radioisotope to be decreased, so that less than the normal 5-10 mCi adult dose can be administered to an adult.
There are several mechanisms by which PDN can improve the use of gallium isotopes, such as Ga-67 (for gamma scintigraphy), for tumor imaging. First, PDN selectively augments a Tf-independent uptake of gallium, and since tumors appear to accumulate gallium by this route to a greater extent than normal tissues, PDN could improve the localization of gallium selectively in tumors. Even if PDN stimulates uptake of gallium in normal tissues as well as tumors, it still has significant beneficial effect in decreasing the necessary interval between time of injection of the radio-tracer and time of imaging. Improving the efficiency of uptake of gallium in tumors or other tissues allows diagnostic images to be obtained at a lower dose of radioactivity to the patient. Tumor specific enhancement of gallium uptake by PDN improves the use of stable gallium as an adjunct to conventional chemotherapy, and concentration of unstable gallium isotopes in tumors for the purpose of administering local radiotherapy.
The present invention therefore includes exposing cells, tissues or tumors to a sufficient dose of the PDN products, for a sufficient period of time, to improve the uptake of gallium into the cells or tumors. The cells can be exposed to the PDN in vitro (for example is an assay) by providing the photo-degradation products (or biological precursors) in a surrounding medium. Alternatively, the PDN can be administered to cells, tissues or tumors in vivo to achieve a systemic blood level, or a local concentration in a tissue of interest (such as a tumor), sufficient to increase gallium uptake in that tissue. Either the PDN products themselves can be administered, or a biological precursor (such as nifedipine) can be administered and allowed to degrade. The degradation may occur by normal metabolic pathways to one of the photo-degradation products. However, the degradation may alternatively be induced by exposure to light, such as pre-irradiation of a solution of nifedipine prior to its administration, or use of light delivered to the tissue of interest (for example through external or endoscopic fiberoptic light delivery of the kind used in photodynamic therapy).
Nifedipine is well-absorbed orally and achieves peak plasma levels approximately 30 minutes post ingestion. In humans treated with nifedipine, a typical dose range is 0.5-2.0 mg/kg/day, given orally in three equally-divided daily doses. It is anticipated that PDN will be similarly well-tolerated and well-absorbed orally, although it may also prove effective if given by other routes, such as by intravenous, subcutaneous or intramuscular injection. PDN is likely to be effective in a dose range similar to that for nifedipine to achieve a local tissue concentration in the range of 0.25-25 xcexcM. Even higher tissue concentrations can be used, because the PDNs are relatively otherwise pharmacologically inert. In vitro, cells which are exposed to the PDN compounds in this concentration range for as little as 10 seconds show enhanced gallium uptake.
Any number of the individual PDN structures, such as those shown in FIG. 1, may demonstrate activity in promoting gallium uptake. These particular PDN products can include nitroso-nifedipine, dehydro-nifedipine, the cis or trans-azoxy nifedipine derivative, the trans-N,Nxe2x80x2-dioxide nifedipine derivative, the hydroxylamine, amine or lactam derivatives, or any other degradation products of nifedipine or other dihydropyridine that increases the uptake of gallium into cells. The cells which are exposed to these compounds are, for example, tumor cells. However, the method of the present invention can also be used with other cells or tissues in vivo in which concentration of gallium is increased by exposure to nifedipine photo-degradation products.
The invention also includes pharmaceutical compositions of nifedipine photo-degradation products or their precursors, either in isolation or in combination with a pharmaceutical carrier, and in unit dosage forms. All routes of administration of PDN products or their precursors are included in this invention. The invention also includes methods of diagnosis and treatment in which nifedipine (or another 4-phenyldihydropyridine derivative) is intentionally exposed to light (such as visible or ultraviolet light) to produce the photo-degradation products. This intentional exposure can take place either prior to administration of the drug to a subject, or in situ in the body. The period of exposure of the nifedipine to light is for a sufficient period of time to produce an adequate concentration of photo-degradation products, for example at least about 1 minute, or 1 to 5 minutes, or even several hours, for example about 4 hours, or as long as a day or more. This invention also includes pharmaceutical compositions of photo-derivatives of nifedipine that are used to promote gallium uptake, regardless of whether these derivatives are produced by photo-irradiation or by alternate methods, such as chemical synthesis.
In particular embodiments, the invention includes a method of increasing gallium uptake by a cell, by exposing the cell to an effective amount of a gallium uptake enhancer comprising a nifedipine photodegradation product, or an analog thereof, that promotes gallium uptake by the cell. The cells are (simultaneously or substantially concurrently) exposed to a gallium compound such as a salt containing a stable or unstable isotope. The gallium compound may be, for example, gallium nitrate, gallium citrate or gallium chloride. Examples of the gallium metal or isotope are Ga-67, Ga-68, GA-69, Ga-71 and Ga-72 (where Ga-69 and 71 are stable isotopes, and the others are unstable radioactive isotopes).
The cell which is exposed to the gallium and the uptake enhancer may be a tumor cell, so that uptake of chemotherapeutic amounts of gallium into the tumor can be differentially increased, compared to non-tumor cells. The cells can also substantially simultaneously be exposed to adjuvant chemotherapeutic anti-neoplastic pharmaceutical agents, such as vinblastine, ifosfamide, hydroxyurea, paclitaxel, cisplatin, methotrexate, 1-beta-D-arabinofuranosylcytosine, and etoposide. Particular examples of tumor cells which could be exposed to the gallium and uptake enhancer are a sarcoma, myeloma, renal adenocarcinoma, testicular leydig cell tumor, medullary thyroid carcinoma, neuroblastoma, melanoma, colon adenocarcinoma, lung adenocarcinoma, or intraductal breast carcinoma.
In yet other embodiments, the method is used to increase uptake of gallium into bone, for example to treat bone specific conditions such as osteoporosis, or to treat hypercalcemia (such as hypercalcemia caused by hyperparathyroidism or malignancy), or to treat Paget""s disease of bone.
The disclosed methods can be used to increase cellular gallium uptake either in vitro or in vivo. For in vivo applications, the gallium and the gallium uptake enhancer are administered to a subject, such as someone who has been diagnosed with a tumor. The gallium may be administered in a therapeutically effective antineoplastic amount, when combined with the gallium uptake enhancer. Alternatively, the gallium may be administered in an amount effective to image the tumor in a gallium scan, when the gallium is administered in combination with the gallium uptake enhancer. Combined administration does not require simultaneous administration, but can refer to simultaneous, substantially simultaneous or separate administration. In particular embodiments, the gallium uptake enhancer is administered prior to the gallium, but within a sufficient period of time to enhance uptake by the tissue of interest (such as the tumor).
Disclosed embodiments of the invention include a gallium uptake enhancer which enhances gallium uptake by a transferrin independent mechanism. Particular examples of such enhancers include a nitrosophenylpyridine, such as the 2xe2x80x2-nitrosophenyl photodegradation product of nifedipine, or a 2xe2x80x2- or 4xe2x80x2-analog thereof. The 2xe2x80x2-nitroso-nifedipine photodegradation product (labeled xe2x80x9cnitroso-derivativexe2x80x9d in FIG. 1) is believed to be particularly effective in promoting gallium uptake.
In yet other embodiments, the gallium uptake enhancer is selected from the group consisting of:
A-B and 
wherein A is a pyridine and B is a nitrosophenyl (such as a 2xe2x80x2-nitrosophenyl or 4xe2x80x2-nitrosophenyl), and n=1-10. Alternatively, the gallium uptake enhancer is: 
wherein
R1-4, R6, and R8-9 are independently selected from the group consisting of H, halogen (particularly Cl), haloalkyl (particularly CCl2), NO2, NO, SO2, a C1-6 alkyl, a COOR10 wherein R10 is H or C1-6 alkyl, and an xe2x80x94OR11 wherein R11 is H or C1-6 alkyl;
and R5 and R7 are independently selected from the group consisting of H, halogen, haloalkyl, NO2, NO, SO2, a C1-6 alkyl, a COOR10 wherein R10 is H or C1-6 alkyl, and an xe2x80x94OR11 wherein R11 is H or C1-6 alkyl, wherein at least one of R5 and R7 is NO.
In particular embodiments in which one of R5 and R7 is NO, R1-9 are selected from the group of H, a C1-6 alkyl, and COOR10, where R10 is lower alkyl, such as methyl or ethyl. In some embodiments, R1xe2x95x90R2xe2x95x90lower alkyl such as methyl, and R4xe2x95x90R5xe2x95x90an ester, such as COOCH3. R5 may be NO, and R6-9xe2x95x90H.
In particular embodiments, R1-4, R6, and R8-9 are independently selected from the group consisting of C1-6 alkyl, a COOR10 wherein R10 is H or C1-6 alkyl, and xe2x80x94OR11 wherein R11 is H or C1-6 alkyl. In even more particular embodiments, R5 is NO and R7 is H; R1xe2x95x90R2xe2x95x90H, R3xe2x95x90R4xe2x95x90COOCH3; and R6xe2x95x90R8xe2x95x90R9xe2x95x90H.
Even more broadly, the gallium uptake enhancer may be selected from 
wherein n=1-10, and
wherein R1-4, R6, and R8-9 are independently selected from the group consisting of H, halogen, haloalkyl, NO2, NO, SO2, a C1-6 alkyl, a COOR10 wherein R10 is H or C1-6 alkyl, and an xe2x80x94OR11 wherein R11 is H or C1-6 alkyl;
and R5 and R7 are independently selected from the group consisting of H, halogen, haloalkyl, NO2, NO, SO2, a C1-6 alkyl, a COOR10 wherein R10 is H or C1-6 alkyl, and an xe2x80x94OR11 wherein R11 is H or C1-6 alkyl, wherein at least one of R5 and R7 is NO.
or wherein
R1-4, R6, and R8-9 are independently selected from the group consisting of H, NO, a C1-6 alkyl, a COOR10 wherein R10 is H or C1-6 alkyl, and an xe2x80x94OR11 wherein R11 is H or C1-6 alkyl;
and R5 and R7 are independently selected from the group consisting of H, NO, a C1-6 alkyl, a COOR10 wherein R10 is H or C1-6 alkyl, and an xe2x80x94OR11 wherein R11 is H or C1-6 alkyl, wherein at least one of R5 and R7 is NO, particularly R5.
Particular embodiments of the method include imaging a tumor with a gallium scan, by administering to a subject an effective amount of a gallium uptake enhancer, such as a nifedipine photodegradation product, or an analog thereof, that increases uptake of gallium by a tumor. A sufficient amount of gallium is also administered to the subject to perform the gallium scan, wherein the sufficient amount of gallium is less than required to perform the gallium scan in the absence of the gallium uptake enhancer. When the gallium uptake enhancer is a transferrin independent gallium uptake enhancer such as a 2xe2x80x2-nitrosophenylpyridine, transferrin independent uptake selectively concentrates the gallium in the tumor to improve the imaging signal obtained from the tumor. When the method is used to improve imaging of tumors, Ga-67 is a particularly suitable isotope, and 50% or less of the usual dose of 10 millicuries of gallium can be administered to perform the scan. Hence a dose of less than about 5 millicuries of the Ga-67 can be used. The uptake enhancer can also allow the tumor to be imaged much more quickly than in the absence of the enhancer. Hence instead of waiting 36-72 hours to obtain the image, the diagnostic procedure can be performed 24 hours or less after administration of the gallium.
In embodiments in which a nifedipine photodegradation product (such as 2xe2x80x2-nitrosophenylpyridine derivative) is administered to the subject, a dose of about 0.5 to about 2.0 mg/kg/day of the nifedipine photodegradation product may be employed. However, the nifedipine photodegradation products are not known to have any biological effect (other than enhancing gallium uptake). In particular, they do not act as calcium channel antagonists. Hence even much higher doses of nifedipine photodegradation products can be used.
In yet other embodiments in which a cutaneous tumor (such as a melanoma) is to be treated, the gallium uptake enhancer is nifedipine applied to skin in an area of the cutaneous tumor, which area is subsequently irradiated with light (such as visible/fluorescent light) that produces the nifedipine photodegradation product gallium uptake enhancer. However, cutaneous and other types of tumors may also be sensitized by administering the gallium uptake enhancer systemically (for example intravenously or orally) to a subject having the tumor.
The invention also includes methods of screening for a gallium uptake enhancer, by exposing cells to a test agent such as a nifedipine photodegradation product, or an analog thereof, in the presence of gallium. The uptake of gallium in the cell is then measured to determine whether the cellular uptake of gallium is greater or less than in the absence of the test agent. In particular disclosed embodiments, the cells are cultured Chinese Hamster Ovary (CHO) cells, such as transferrin receptor negative CHO cells.
Additional objects and advantages of the present invention will be apparent from the following detailed description of a preferred embodiment, which proceeds with reference to the accompanying figures.